1. Field of Inventive Subject Matter
The inventive subject matter relates to novel methods for treating T-cell deficiencies using MHC-disparate T-cell precursors derived from universal donors in ‘off-the-shelf’ immunotherapy methods. For example, the inventive subject matter comprises treating individuals suffering from such conditions as T-cell-depletion following irradiation injury, T-cell-depletion following cytostatic therapy, and other diseases, disorders, and conditions resulting in T-cell-depletion. The inventive methods can be further enhanced by genetic engineering for targeted immunotherapy.
2. Background
Hematopoietic cell transplantation is the transplantation of blood stem cells derived from the bone marrow or blood of the donor, most often performed for people with diseases of the blood, bone marrow, or certain types of cancer.
Hematopoietic cell transplantation remains a risky procedure with many possible complications; it has heretofore been reserved for patients with life-threatening diseases.
T-Cell Deficiencies.
T-cell deficiencies can occur in many physiological and pathophysiological settings. Thymic involution during aging is an important cause of thymic atrophy resulting in impaired T-cell function (see, for example, Mackall, C. L. & Gress, R. E. Thymic aging and T-cell regeneration. Immunol Rev 160, 91-102 (1997)). Various autoimmune disorders, genetic diseases, hematological malignancies, and infectious diseases are all associated with defective T-cell immunity (see, for example, Grunebaum, et al., Human T cell immunodeficiency: when signal transduction goes wrong. Immunol Res 35, 117-126 (2006); Fischer, et al., Naturally occurring primary deficiencies of the immune system. Annu Rev Immunol 15, 93-124 (1997); and Chinen, et al., Advances in basic and clinical immunology. J Allergy Clin Immunol 118, 489-495 (2006)).
Therapy-induced and unintentional exposures to cytostatic or cytotoxic agents, such as chemotherapeutics or gamma-irradiation, frequently cause transient or long-lasting T-cell deficiencies (see, for example, Lehrnbecher, et al., Therapy-induced alterations in host defense in children receiving therapy for cancer. J Pediatr Hematol Oncol 19, 399-417 (1997) and Yarilin, et al., Late T cell deficiency in victims of the Chemobyl radiation accident: possible mechanisms of induction. Int J Radiat Biol 63, 519-528 (1993)).
Hematopoietic Stem Cell Transplantation.
There are two primary types of hematopoietic stem cell transplantation (“HSCT”). Autologous HSCT involves isolation of hematopoietic stems cells (HSC) from the patient and storage of the harvested cells. The patient is then treated with high-dose chemotherapy with or without radiotherapy in the form of total body irradiation, to eradicate the patient's malignant cell population. This takes place at the cost of also eliminating the patient's bone marrow stem cells. In autologous HSCT, the patient's own stored stem cells are then returned to their body. Autologous transplants have the advantage of a lower risk of graft rejection and infection, since the recovery of immune function is rapid. The incidence of a patient experiencing graft-versus-host disease is close to none as the donor and recipient are the same individual.
Allogeneic HSCT involves two people, one is the (healthy) donor and one is the (patient) recipient. Allogeneic HSC donors must have a tissue (HLA) type that matches the recipient. Matching is performed on the basis of variability at three or more loci of the (HLA) gene, and a perfect match at these loci is preferred. Even if there is a good match at these critical alleles, the recipient will require immunosuppressive medications to mitigate graft-versus-host disease. Allogeneic transplant donors may be related (usually a closely HLA matched sibling) or unrelated (donor who is not related and found to have very close degree of HLA matching). Allogeneic transplants are also performed using umbilical cord blood as the source of stem cells.
Many recipients of HSCTs are leukemia patients who would benefit from treatment with high doses of chemotherapy or total body irradiation. Other conditions treated with stem cell transplants include sickle-cell disease, myelodysplastic syndrome, neuroblastoma, lymphoma, Ewing's Sarcoma, Desmoplastic small round cell tumor, Hodgkin's disease, and multiple myeloma. More recently non-myeloablative, or so-called “mini transplant,” procedures have been developed that require smaller doses of preparative chemo and radiation.
HSCT is associated with a fairly high mortality in the recipient (10% or higher), which limits its use to conditions that are themselves life-threatening. Major causes of complications are veno-occlusive disease, mucositis, infection (sepsis) and graft-versus-host disease. HSCT is also associated with a particularly prolonged defect in T-cell function, yet the immunotherapeutic activity of an HSCT procedure is critical for its overall anti-tumor effect (see, for example, Appelbaum, Haematopoietic cell transplantation as immunotherapy. Nature 411, 385-389 (2001)). In the allogeneic setting, T-cells are primarily responsible for the negative effect of graft-versus-host disease (GVHD) and the therapeutic benefit of graft-versus-tumor (GVT) activity. Optimizing graft-versus-tumor activity while minimizing graft-versus-host disease is one of the major challenges in such transplants.
Graft-versus-host disease.
Graft-versus-host disease (GVHD) is an inflammatory disease that is unique to allogeneic transplantation. It is an attack by transplanted leukocytes against the recipient's tissues. This can occur even if the donor and recipient are HLA-identical, because the immune system can still recognize other differences between tissues. It is aptly named graft-versus-host disease because bone marrow transplantation is the only transplant procedure in which the transplanted cells must accept the body rather than the body accepting the new cells. Acute graft-versus-host disease typically occurs in the first 3 months after transplantation and may involve the skin, intestine, or the liver. Corticosteroids such as prednisone are a standard treatment.
Chronic graft-versus-host disease may also develop after allogeneic transplant and is the major source of late complications. In addition to inflammation, chronic graft-versus-host disease may lead to the development of fibrosis, or scar tissue, similar to scleroderma or other autoimmune diseases and may cause functional disability, and the need for prolonged immunosuppressive therapy. Graft-versus-host disease is usually mediated by T cells when they react to foreign peptides presented on the MHC of the host. Removal of these T cells before donation can lessen the risk of this disease.
T-Cell Based Therapy.
T-cell based therapies such as donor leukocyte infusion or protocols involving ex vivo expansion and manipulation of T-cells in order to generate tumor or virus-specific cells have been used for many years as strategies to enhance immune reconstitution and anti-tumor activity following hematopoietic stem cell transplantation. However, these therapies are associated with a variety of problems including the following:                a. limited availability of suitable cells because T-cells have to be either autologous or MHC-matched allogeneic;        b. contamination with residual malignant T-cells when using autologous cells;        c. graft-versus-host disease when using allogeneic cells;        d. in vivo, cytokine administration is required during hematopoietic stem cell transplantation; and        e. adoptively transferred cells have a short life span.        
Zakrzewski, et al., Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation, Nat. Med., 12(9):1039-47 (2006), is fairly indicative of the general understanding of skilled artisans in the relevant art, that the use of adoptive T-cell therapies requires co-administration of HSCs.
United States Patent Publication No. 2004/0067583 (Bernstein, et al.), requires either an autograft of T-cell precursors or an allograft of T-cell precursors which requires co-administration of immunosuppressives and co-administration of allogeneic HSCs, and fails to teach or suggest the use of CD4− CD8− double negative (DN) precursor cells.
United States Patent Publication No. 2004/0171148 (Schmitt, et al.), requires either an autograft of T-cell precursors or an allograft of T-cell precursors which requires co-administration immunosuppressives and co-administration of allogeneic HSCs.
Thus, the use of adoptive T-cell therapies is often limited by barriers imposed by MHC disparity. There is a long-felt and unmet need in the art to develop a way to address T-cell deficiencies without rejection, alloreactivity, and impaired antigen presentation, and to identify a universal donor strategy which does not require long term administration of immunosuppressives to graft recipients. The inventive subject matter provides methods which reduce or eliminate these problems, for the first time demonstrating that lymphoid precursor cells from a non-MHC-matched universal donor can be successfully transferred to any individual, irrespective of MHC disparities. Applicants have demonstrated that allogeneic T-cell precursors, when adoptively transferred to irradiated recipients, without syngeneic hematopoietic stem cells, develop into functional mature T-cells.
Surprisingly, Applicants have found that allogeneic T-cell precursors are effective when used alone for adoptive transfer across MHC barriers, even in the absence of allogeneic hematopoietic stem cells, and are able to overcome T-cell deficiencies such as injury resulting from exposure to radiation and diminished T-cell function. Further, in individuals undergoing cancer treatment and others with T-cell deficiencies, allogeneic T cell precursor transfer can improve anti-tumor activity in immunosuppressed recipients.
Thus, the inventive subject matter provides novel methods for therapy using adoptively transferred ex vivo generated allogeneic T-cell precursors that can develop into host-MHC restricted T-cells characterized by dual tolerance and selection of a functional TCR repertoire, even in a fully mismatched thymic epithelial MHC environment. Specifically, the primary advantages of utilizing T-cell precursors for immunotherapy are the following: (1) T-cell precursors do not have to be MHC matched, since graft-versus-host disease is not an issue; (2) the use of allogeneic precursors cells instead of autologous cells eliminates the risk of contamination with residual malignant autologous cells; and (3) the generation and storage of virtually unlimited quantities of precursor cells from universal donors for ‘off-the-shelf’ immunotherapy is thus achieved.
The inventive methods have these substantial logistic and technical advantages, and additionally facilitate the use of ex vivo manipulation protocols, in particular genetic engineering, to generate target-antigen-specific or otherwise enhanced designer cells. Adoptive transfer of MHC mismatched and genetically enhanced T-cell precursors therefore represents a novel, labor-saving, and cost-effective process for targeted ‘off-the-shelf’ immunotherapy for patients with a malignant disease or other T-cell deficiency.